Human cytomegalovirus (HCMV), a member of the herpesviridae family of viruses, is a significant human pathogen, causing pneumonitis, blindness, and death among transplant and AIDS patients and mental retardation and hearing loss among congenitally infected newborns. Currently available antivirals target the viral DNA polymerase but suffer from significant toxicities. HCMV encodes a protein, pUL98 that has both endonuclease and 5'-3' exonuclease activities in vitro. The function of pUL98 in HCMV replication is not known; however, studies of homologous nucleases expressed by other herpesviruses have suggested that they may function to debranch complex concatemeric DNA replicative intermediates prior to DNA insertion into capsids. Similarities to phage l reda (exonuclease) and the ability to promote strand-exchange reactions mediated by viral single-stranded DNA binding protein further suggest a potential role in recombination, a key feature of herpesvirus DNA replication. Recent genetic studies suggest that the UL98 gene is essential for viral replication; pUL98 therefore presents an attractive target for antiviral interventions. In aim 1 of this proposal we will identify active site amino acids critical for the in vitro enzymatic activities of pUL98 using mutagenesis and recombinant protein expression. In aim 2 we will determine the importance of these activities for viral replication by construction of mutant viral genomes in which pUL98 enzymatic activities are disabled by selective active site mutations. Through these studies we will determine if (1) pUL98 is essential for HCMV replication and (2) if it is pUL98's nucleolytic activities that are crucial. Demonstration that an enzymatic activity of pUL98 is important for HCMV replication will provide strong justification for further studies to identify compounds that selectively inhibit the activity, either through determination of the enzyme active site structure and custom drug design or through high throughput screening of compound libraries using in vitro enzymatic assays. Such studies could lead to novel pUL98 inhibitors that could be clinically useful in treating HCMV infections alone or in combination with existing DNA polymerase inhibitors. Human cytomegalovirus is the major infectious cause of birth defects in the United States and is an important pathogen in AIDS and transplant patients. The proposed studies will investigate the potential of the viral alkaline nuclease (pUL98) as a target for the development of novel antivirals. By evaluating the impact on viral replication of genetically inactivating the enzymatic activities of alkaline nuclease, the potential impact of pharmacological inhibitors can be predicted. Demonstration that these activities are important for viral replication would provide strong justification for future efforts towards discovery of compounds that target this protein's activities. [unreadable] [unreadable] [unreadable]